Treatment of Hearing Loss by Inhibition of Casein Kinase 1

ABSTRACT

Methods for treating hearing loss that include administering an inhibitor, e.g., a small molecule inhibitor, of casein kinase 1, preferably in combination with a treatment that stimulates Atoh1 gene expression, e.g., a gamma-secretase inhibitor, an Atoh1 stimulatory compound, or a GSK-3-beta inhibitor.

CLAIM OF PRIORITY

This application is a divisional of U.S. patent application Ser. No. 15/781,041, filed on Jun. 1, 2018, which is a 371 U.S. National Phase Application of PCT/US2016/064727, filed on Dec. 2, 2016, which claims the benefit of U.S. Provisional Patent Application Ser. No. 62/263,412, filed on Dec. 4, 2015. The entire contents of the foregoing are hereby incorporated by reference herein.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No. RO1 DC007174 awarded by the National Institutes of Health. The Government has certain rights in the invention.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submitted electronically as an ASCII text file named “Sequence_Listing.txt.” The ASCII text file, created on Oct. 8, 2021, is 8 KB in size. The material in the ASCII text file is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

Described herein are methods for treating hearing loss that include administering an inhibitor, e.g., a small molecule inhibitor, of casein kinase 1, preferably in combination with a treatment that stimulates Atoh1 gene expression, e.g., a gamma-secretase inhibitor, an Atoh1 stimulatory compound, or a GSK-3-beta inhibitor.

BACKGROUND

Loss of mammalian cochlear hair cells, caused by genetic mutations, autoimmune disease, ototoxic medications, exposure to noise, and aging, is usually permanent and can lead to mild to complete hearing loss in affected subjects.

SUMMARY

The present disclosure provides, inter alia, methods and pharmaceutical compositions for treating subjects for the conditions described herein. Accordingly, the present disclosure is based, at least in part, on the discovery that differentiation of a cell to or towards a mature cell of the inner ear, e.g., an auditory hair cell can be promoted through β-catenin-dependent WNT signaling. In other words, the present disclosure provides methods and compositions relating to the WNT/β-catenin signaling pathway for generating cells that have characteristics of auditory hair cells.

Thus, the present disclosure provides methods for treating a subject who has or is at risk of developing hearing loss or vestibular dysfunction. The methods can include identifying a subject who has experienced, or is at risk for developing, hearing loss or vestibular dysfunction; administering to the subject, e.g., to the ear of the subject one or more one or more casein kinase 1 (CK1) inhibitors, and optionally one or more compounds that stimulate Atoh1 gene expression, e.g., a gamma-secretase inhibitor, an Atoh1 stimulatory compound, or a GSK-3-beta inhibitor, thereby treating the hearing loss or vestibular dysfunction in the subject. Also provided herein are a casein kinase 1 (CK1) inhibitor, and optionally a compound that stimulates Atoh1 gene expression, for use in treating a subject who has or is at risk of developing hearing loss or vestibular dysfunction.

In some embodiments, the subject has or is at risk for developing sensorineural hearing loss, auditory neuropathy, or both.

In some embodiments, the subject has or is at risk for developing a vestibular dysfunction that results in dizziness, imbalance, or vertigo.

In some embodiments, the casein kinase 1 (CK1) inhibitors and/or the one or more compounds that stimulate Atoh1 gene expression is administered systemically. In some embodiments, the casein kinase 1 (CK1) inhibitors and/or the one or more compounds that stimulate Atoh1 gene expression is administered locally to the ear of the subject, e.g., to the inner ear. In some embodiments, the one or more compounds that stimulate Atoh1 gene expression comprises one or more glycogen synthase kinase 3 β (GSK3β) inhibitors. In some embodiments, the one or more small molecule casein kinase 1 (CK1) inhibitor is D4476. In some embodiments, the one or more compounds that stimulate Atoh1 gene expression is a gamma secretase inhibitor. In some embodiments, a combination of CHIR99021 and LY411575 is used.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1. CK1 binds to Atonal homolog 1 (Atoh1) and decreases steady-state Atoh1 level

HEK cells were co-transfected with FLAG Atoh1 plasmid and HA-CK1 plasmids (including CK1α, CK1δ, and CK1ε). Immunoprecipitation was performed under denaturing conditions with an anti-FLAG antibody. Ck1 was detected with an anti-Myc antibody while Atoh1 was detected with anti-FLAG antibody.

FIG. 2. CK1 inhibitor diminishes Huwe1 binding and decreases ubiquitylation of Atoh1

HEK cells were co-transfected with HA-ubiquitin and FLAG-Atoh1 plasmids for 48 hours and treated with CK1 inhibitors (PF-670462 or D4476) and/or proteasome inhibitor MG132 (10 μM) for 4 hours. Immunoprecipitation was performed under denaturing conditions with anti-FLAG antibody. Endogenous Huwe1 was detected with an anti-Huwe1 antibody. Atoh1 was detected with anti-FLAG while ubiquitin was detected with anti-HA antibodies.

FIGS. 3A-B. Evolutionarily conserved serines in the C-terminus of Atoh1 account for its stability

(A) Half-life analysis of truncated Atoh1 over a 4-hour time frame. HEK cells were transfected with either wild-type or truncated FLAG Atoh1 for 48 hours and incubated with cycloheximide (100 μg/ml) for the indicated times. β-actin served as a loading control for input protein.

(B) Quantification of protein half-lives. The ratio of Atoh1 to β-actin based on densitometry was plotted.

FIGS. 4A-B. A signal for Huwe1 binding is located in the C-terminus of Atoh1

(A) Truncation of Atoh1 influences Huwe1 interaction. HEK cells were co-transfected with HA-ubiquitin and wild-type or truncated FLAG Atoh1 plasmids (Δ10-93 for deletion 1, Δ94-156 for deletion 2, Δ214-305 for deletion 3 and Δ306-347 for deletion 4) for 48 hours. Immunoprecipitation was performed under denaturing conditions with anti-FLAG antibody. Atoh1 was detected with the anti-HA and anti-FLAG antibodies. Endogenous Huwe1 was detected with an anti-Huwe1 antibody. Immunoprecipitation with IgG was used for the control.

(B) Blotting of endogenous Huwe1 and Atoh1. Five percent of total extracts from the experiment shown in A were analyzed by Western blotting with an anti-FLAG antibody to detect Atoh1 and anti-Huwe1 to detect endogenous Huwe1.

FIGS. 5A-C. Serine 334 is a critical residue for Atoh1 degradation

(A) C-terminal regions of Atoh1 (area 4) of different species were aligned. Conserved serines at 325, 328, 331 and 334 are marked with asterisks. The corresponding residues in the human sequence are 328, 331, 334 and 337. The sequences shown are SEQ ID NOs:12-19 in order, respectively.

(B) Serine to alanine mutations affects the steady-state level of Atoh1. HEK cells were transfected with wild-type or mutated FLAG Atoh1 plasmids for 40 hours and treated with either vehicle (DMSO) or MG132 (10 μM). After treatment with proteasome inhibitor for 6 hours, S334A had the smallest increase in Atoh1 (vehicle treatment is marked with a minus sign) compared to wild-type or other Atoh1 mutants.

(C) Half-life analysis of mutated Atoh1 proteins over a 4-hour time frame. HEK cells were transfected with either wild-type or mutated FLAG Atoh1 plasmids for 40 hours and incubated with cycloheximide (100 μg/ml) for the indicated times. The ratio of Atoh1 to β-actin based on densitometry was plotted.

FIG. 6. Mutation at Serine 334 of Atoh1 decreases Huwe1 binding and ubiquitylation

Mutation of Atoh1 influences Huwe1 interaction. HEK cells were co-transfected with ubiquitin with all lysines except K48 mutated and wild type or mutated FLAG Atoh1 plasmids (S334A or S328/329A) for 48 hours. Immunoprecipitation was performed under denaturing conditions with an anti-FLAG antibody. Endogenous Huwe1 was detected with an anti-Huwe1 antibody. Atoh1 was detected with an anti-FLAG antibody while ubiquitin was detected with an anti-HA antibody.

FIGS. 7A-C. 5334 is essential for CK1-mediated Atoh1 downstream signaling

Dual luciferase assay using a firefly reporter construct with an AtEAM motif in HEK cells. Experiments were done in triplicate, and data are presented as the mean±SEM after normalization to Renilla luciferase

(A) CK1 inhibition (D4476) increases Atoh1-specific E-box activity. DMSO or CK1 inhibitor D4476 was added to HEK with or without overexpression of Atoh1 for overnight to block the degradation of Atoh1 and therefore downstream Atoh1-specific E-box activity.

(B) CK1δ and/or CK1c reduce Atoh1-specific E-box activity. Overexpression of CK1δ and/or CK1c in HEK cells co-transfected with Atoh1 plasmid significantly reduce Atoh1-specific E-box activity. The effect of CK1 isoform, CK1a, was not significant

(C) S334 is essential for CK1-mediated reduction of Atoh1-specific E-box activity. HEK cells were co-transfected with CK1δ & CK1c (CK1c/8) and wild-type or mutated Atoh1 plasmids. CK1-mediated reduction of Atoh1-specific E-box activity was abolished when S334 residue was mutated to alanine (S334A). Such CK1-mediated reduction was seen for other mutated residues, including S325A, S328A, S331A and S339A.

FIG. 8. CK1 inhibition stabilizes Atoh1 protein in the organ of Corti.

Effect of CK1 inhibition on Atoh1 upregulation in the organ of Corti. Newborn organs of Corti (P1) were treated with the CK1 inhibitor D4476 (10 uM) for 72 hours. Atoh1 were quantified after Western blotting by densitometry and normalized to a loading control (beta-actin).

FIGS. 9A-C. Sox2 Lineage Tracing of Supporting Cells in Neonatal Cochleae Treated with Ck1 inhibitor D4476.

(A) Double-labeled cells positive for Sox2 lineage (tomato) and myosin VIIa (green) were found both in the inner hair cell and outer hair cell area in the mid-base region of cochlear tissue from neonatal mice carrying the Sox2-CreER as well as the Cre reporter 3 days after treatment with CK1 inhibitor D4476 (10 nM). Hair cell co-labeling with the lineage tag indicates derivation from a Sox2-positive cell (labeled with asterisks) and is thus evidence for newly generated hair cells by transdifferentiation of supporting cells. OHCs, at the bottom of the image, are delineated by a light gray bracket while IHCs, at the top of the image, are delineated by a white bracket. The scale bar is 25 μm.

(B, C) Effect of CK1 inhibition was significant. Quantification of the reporter-positive OHC and IHC counts for Sox2 lineage tracing of DMSO and D4476 treated explants showed significantly more reporter-labeled OHCs (B) and IHCs (C) across most cochlear regions after D4476 treatment (mean±SEM per 100 mm, plotted on a logarithmic scale; *p<0.05, **p<0.01, n=4-8 for both groups).

FIG. 10. Transdifferentiation of Sox2-positive supporting cell into hair cells results from CK1 inhibition after aminoglycoside damage.

Images of the basal turn of the lineage-traced organ of Corti damaged by gentamycin and treated with CK1 inhibitor D4476 for 72 hours. Doubled-labeled cells positive for Sox2 lineage (tomato in original image) and myosin VIIa (green in original image), marked with an asterisk in the leftmost panel, indicated transdifferentiation of supporting cells. The scale bar is 25 μm.

DETAILED DESCRIPTION

Mammals show limited ability to regenerate hair cells (Forge et al., Science 259, 1616-1619 (1993); Warchol et al., Science 259, 1619-1622 (1993)). Hair cell differentiation is dependent on basic helix-loop-helix (bHLH) transcription factor, Atoh1. Overexpression of Atoh1 via gene transfer results in the generation of new hair cells from inner ear progenitors in the organ of Corti (Bermingham et al., Science 284, 1837-1841 (1999); Jeon et al., Mol Cell Neurosci 34, 59-68 (2007)). Several regulatory pathways have been found to be involved in Atoh1 regulation (Zhang et al., Stem Cells 31(12):2667-79 (2013); Jeon et al., J. Neurosci. 31, 8351-8358 (2011); Kelley et al., Nat Rev Neurosci 7, 837-849 (2006); Shi et al., J Biol Chem 285, 392-400 (2010)). The present inventors have discovered that posttranslationally, levels of Atoh1 are controlled at least in part by ubiquitylation by Huwe1, a HECT-domain, E3 ubiquitin ligase, which targets Atoh1 for proteasomal degradation by polyubiquitylation (see Cheng, “Role of the ubiquitin-proteasome pathway in the inner ear: identification of an E3 ubiquitin ligase for Atoh1,” Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2014, available online at hdl.handle.net/1721.1/96458; and PCT/US2015/043976).

In addition to E3 ubiquitin ligase, specification of a substrate for ubiquitylation and degradation also comes from post-translational modification of degrons (a sequence within a protein that is sufficient for recognition and degradation by a proteolytic apparatus), which allows substrate ubiquitylation in response to endogenous or external signals (Ravid and Hochstrasser, Nat Rev Mol Cell Biol. 9(9):679-90 (2008)). We found that the serine-enriched C-terminus of Atoh1 had a number of evolutionarily conserved serines at positions 309, 325, 328, 331 and 334, and putative motifs for phosphorylation by casein kinase 1 (CK1), pSer/Thr-X-XSer/Thr, starting from Ser 325, followed by Ser 328, 331 and 334. CK1 is a serine/threonine protein kinase that triggers phosphorylation of substrates, and has Ck1α, CK1δ, CK1ε and CK1γ isoforms. Since CK1 isoforms have molecular weight ranging from 32 to 52.5 kDa (Knippschild et al., 2014), which are close to Atoh1, we conducted mass spectrometry on the band at 45 kDa from lysates immunoprecipitated with Atoh1 (Table I). CK1ε and CK1γ were identified in the proteins at this molecular weight. Small molecule CK1 inhibitors, including D4476 (4-[4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide), IC-261 (1,3-Dihydro-3-[(2,4,6-trimethoxyphenyl)methylene]-2H-indol-2-one, also known as SU5607), and PF-670462 (4-[3-Cyclohexyl-5-(4-fluoro-phenyl)-3H-imidazol-4-yl]-pyrimidin-2-ylamine dihydrochloride) increased the steady-state abundance of Atoh1 in human 293T cells; D4476 was also shown to increase hair cell generation in the organ of Corti and promote hair cell regeneration after aminoglycoside damage.

In some embodiments, the present methods include using a CK1 inhibitor, e.g., a small molecule CK1 inhibitor, which increases the half-life and thus the overall amount of Atoh1 protein, is administered in combination with a treatment that stimulates Atoh1 gene expression, e.g., a gamma-secretase inhibitor, an Atoh1 stimulatory compound, or a GSK-3-beta inhibitor.

Casein Kinase Inhibitors Casein Kinase 1 inhibitors include, e.g., PF 670462 (4-[1-Cyclohexyl-4-(4-fluorophenyl)-1H-imidazol-5-yl]-2-pyrimidinamine dihydrochloride) (Sigma); IC261 (3-[(2,4,6 trimethoxyphenyl)methylidenyl]-indolin-2-one (Abcam); D 4476 (4-[4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (R &D Systems); LH 846 (N-(5-Chloro-6-methyl-2-benzothiazolyl)benzeneacetamide) (Tocris Bioscience); 6-cycloamino-3-(pyrid-4-yl)imidazo[1,2-b]pyridazine derivatives (Sanofi Aventis), see US20100179154. Casein Kinase 1 selective inhibitors also include the following: 4-((6-methoxy-3-pyridinyl)methylene)-2-(5-fluoro-2-thienyl)-5(4H)-oxazolone; 4-((6-methoxy-3 pyridinyl)methylene)-2-(5-chloro-2-thienyl)-5(4H)-oxazolone; 4-((6-methoxy-3-pyridinyl)methylene)-2-(5-bromo-2-thienyl)-5(4H)-oxazolone; 4-((6-methoxy-3-pyridinyl)methylene)-2-(5-iodo-2-thienyl)-5(4H)-oxazolone (Pfizer), see U.S. Pat. No. 8,518,944; PF-4800567 (3-[(3-Chlorophenoxy)methyl]-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine hydrochloride) (Calbiochem); 4-[4-(4-Fluorophenyl)-5-(2-pyridyl)-1-hydroxy-1H-imidazol-2-yl]benzonitrile and derivatives (Roche), see US20090099237.

In some embodiments, suitable kinase inhibitors can include inhibitory nucleic acids, e.g., shRNA or siRNA, that target CK1 and decrease CK1 protein levels. In some embodiments, suitable kinase inhibitors does not include inhibitory nucleic acids that target CK1 and decrease CK1 protein levels.

Gamma-Secretase Inhibitors

Gamma secretase inhibitors useful in the present methods include, e.g., RO4929097; DAPT (N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester); L-685458 ((5S)-(t-Butoxycarbonylamino)-6-phenyl-(4R)hydroxy-(2R)benzylhexanoyl)-L-leu-L-phe-amide); BMS-708163 (Avagacestat); BMS-299897 (2-[(1R)-1-[[(4-Chlorophenyl)sulfonyl](2,5-difluorophenyl)amino]ethyl-5-fluorobenzenebutanoic acid); MK-0752; YO-01027; MDL28170 (Sigma); LY411575 (N-2((2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl)-N1-((7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)-1-alaninamide, see U.S. Pat. No. 6,541,466); ELN-46719 (2-hydroxy-valeric acid amide analog of LY411575 (where LY411575 is the 3,5-difluoro-mandelic acid amide) (U.S. Pat. No. 6,541,466)); PF-03084014 ((S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-3-ylamino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide, Samon et al., Mol Cancer Ther 2012; 11:1565-1575); and Compound E ((2S)-2-{[(3,5-Diflurophenyl)acetyl]amino}-N-[(3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]propanamide; see WO 98/28268 and Samon et al., Mol Cancer Ther 2012; 11:1565-1575; available from Alexis Biochemicals)), or pharmaceutically acceptable salts thereof.

In some embodiments, suitable gamma secretase inhibitors include: semagacestat (also known as LY450139, (2S)-2-hydroxy-3-methyl-N-[(1S)-1-methyl-2-oxo-2-[[(1S)-2,3,4,5-tetrahydro-3-methyl-2-oxo-1H-3-benzazepin-1-yl]amino]ethyl]butanamide, available from Eli Lilly; WO 02/47671 and U.S. Pat. No. 7,468,365); LY411575 (N-2((2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl)-N1-((7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)-L-alaninamide, available from Eli Lilly, Fauq et al., Bioorg Med Chem Lett 17: 6392-5, 2007); begacestat (also known as GSI-953, U.S. Pat. No. 7,300,951); arylsulfonamides (AS, Fuwa et al., Bioorg Med Chem Lett. 16(16):4184-4189, 2006); N—[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester (DAPT, Shih and Wang, Cancer Res. 67: 1879-1882, 2007); N—[N-3,5-Difluorophenacetyl]-L-alanyl-S-phenylglycine Methyl Ester (also known as DAPM, gamma-Secretase Inhibitor XVI, available from EMD Millipore); Compound W (3,5-bis(4-Nitrophenoxy)benzoic acid, available from Tocris Bioscience); L-685,458 ((5S)-(tert-Butoxycarbonylamino)-6-phenyl-(4R)-hydroxy-(2R)-benzylhexanoyl)-L-leucy-L-phenylalaninamide, available from Sigma-Aldrich, Shearmen et al., Biochemistry 39, 8698-8704, 2000); BMS-289948 (4-chloro-N-(2,5-difluorophenyl)-N-((1R)-{4-fluoro-2-[3-(1H-imidazol-1-yl)propyl]phenyl}ethyl)benzenesulfonamide hydrochloride, available from Bristol Myers Squibb); BMS-299897 (4-[2-((1R)-1-{[(4-chlorophenyl)sulfonyl]-2,5-difluoroanilino}ethyl)-5-fluorophenyl]butanoic acid, available from Bristol Myers Squibb, see Zheng et al., Xenobiotica 39(7):544-55, 2009); avagacestat (also known as BMS-708163, (R)-2-(4-chloro-N-(2-fluoro-4-(1,2,4-oxadiazol-3-yl)benzyl)phenylsulfonamido)-5,5,5-trifluoropentanamide, available from Bristol Myers Squibb, Albright et al., J Pharmacol. Exp. Ther. 344(3):686-695, 2013); MK-0752 (3-(4-((4-chlorophenyl)sulfonyl)-4-(2,5-difluorophenyl)cyclohexyl)propanoic acid, available from Merck); MRK-003 ((3′R,6R,9R)-5′-(2,2,2-trifluoroethyl)-2-((E)-3-(4-(trifluoromethyl)piperidin-1-yl)prop-1-en-1-yl)-5,6,7,8,9,10-hexahydrospiro[6,9-methanobenzo[8]annulene-11,3′-[1,2,5]thiadiazolidine] 1′,1′-dioxide, available from Merck, Mizuma et al., Mol Cancer Ther. 11(9):1999-2009, 2012); MRK-560 (N-[cis-4-[(4-Chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoro-methanesulfonamide, Best et. al., J Pharmacol Exp Ther. 317(2):786-90, 2006); RO-4929097 (also known as R4733, (S)-2,2-dimethyl-N1-(6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)-N3-(2,2,3,3,3-pentafluoropropyl)malonamide, available from Hoffman-La Roche Inc., Tolcher et al., J Clin. Oncol. 30(19):2348-2353, 2012); JLK6 (also known as 7-Amino-4-chloro-3-methoxyisocoumarin, available from Santa Cruz Biotechnology, Inc., Petit et al., Nat. Cell. Biol. 3: 507-511, 2001); Tarenflurbil (also known as (R)-Flurbiprofen, (2R)-2-(3-fluoro-4-phenylphenyl)propanoic acid); ALX-260-127 (also known as Compound 11, described by Wolfe et al., J. Med. Chem. 41: 6, 1998); Sulindac sulfide (SSide, Takahashi et al., J Biol Chem. 278(20): 18664-70, 2003); 1,1,1-trifluoro-N-(4-[5-fluoro-2-(trifluoromethyl)phenyl]-4-{[4(trifluoromethyl)phenyl]sulfonyl}cyclohexyl)methanesulfonamide (described in US20110275719); N-[trans-3-[(4-chlorophenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclobutyl]-1,1,1-trifluoromethanesulfonamide (described in US20110263580); N-[cis-3-[(4-chlorophenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclobutyl]-1,1,1-trifluoromethanesulfonamide (described in US20110263580); N-[cis-3-[(4-chlorophenyl)sulfonyl]-3-(2-cyano-5-fluorophenyl)cyclobutyl]-1,1,1-trifluoromethanesulfonamide (described in US20110263580); N-[cis-3-[(4-chlorophenyl)sulfonyl]-3-(2,5-dichlorophenyl)cyclobutyl]-1,1,1-trifluoromethanesulfonamide (described in US20110263580); N-(cis-3-(2,5-difluorophenyl)-3-{[4-(trifluoromethyl)phenyl]sulfonyl}cyclobutyl)-1,1,1-trifluoromethanesulfonamide (described in US20110263580); N-{cis-3-(5-chloro-2-fluorophenyl)-3-[(4-chlorophenyl)sulfonyl]cyclobutyl}-1,1,1-trifluoromethanesulfonamide (described in US20110263580); N-{cis-3-(2,5-difluorophenyl)-3-[(4-fluorophenyl)sulfonyl]cyclobutyl}-1,1,1-trifluoromethanesulfonamide (described in US20110263580); N-{cis-3-(2,5-difluorophenyl)-3-[(3,4-difluorophenyl)sulfonyl]cyclobutyl}-1,1,1-trifluoromethanesulfonamide (described in US20110263580); N-[cis-3-[(4-cyanophenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclobutyl]-1,1,1-trifluoromethanesulfonamide (described in US20110263580); 4-{[cis-3-[(4-chlorophenyl)sulfonyl]3-(2,5-difluorophenyl)cyclobutyl][trifluoromethyl) sulfonyl]amino}butanoic acid (described in US20110263580); N-[cis-3-[(4-chlorophenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclobutyl]-1,1,1-trifluoro-N-[2-(tetrahydro-2-pyran-2-yloxy)ethyl]methanesulfonamide (described in US20110263580); Methyl{[cis-3-[(4-chlorophenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclobutyl][(trifluoromethyl)sulfonyl]amino}acetate (described in US20110263580); N-[3-[(4-chlorophenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclobutyl]-1,1,1-trifluoro-N-methylmethanesulfonamide (described in US20110263580); N-[3-[(4-chlorophenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclobutyl]-1,1,1-trifluoro-N-methylmethanesulfonamide (described in US20110263580); Methyl 4-{[cis-3-[(4-chlorophenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclobutyl][(trifluoro-methyl)sulfonyl]amino}butanoate (described in US20110263580); N-[cis-3-[(4-chlorophenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclobutyl]-N-[(trifluoromethyl)sulfonyl]glycine (described in US20110263580); N-[cis-3-[(4-chlorophenyl)sulfonyl]-3-(2,5-difluorophenyl)-1-methylcyclobutyl]-1,1,1-trifluoromethanesulfonamide (described in US20110263580); N-(cis-3-(2,5-difluorophenyl)-1-methyl-3-{[4-(trifluoromethyl)phenyl]sulfonyl}cyclobutyl)-1,1,1-trifluoromethanesulfonamide (described in US20110263580); N-[cis-3-[(4-chlorophenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclobutyl]-1,1,1-trifluoro-N-[(trifluoromethyl)sulfonyl]methanesulfonamide (described in US20110263580); Sodium[cis-3-[(4-chlorophenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclobutyl][(trifluoromethyl)sulfonyl]azanide (described in US20110263580); Potassium[cis-3-[(4-chlorophenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclo butyl][(trifluoromethyl)sulfonyl]azanide (described in US20110263580); N-[cis-3-[(4-trifluoromethoxyphenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclobutyl]-1,1,1-trifluoromethanesulfonamide (described in US20110263580); 1,1,1-trifluoro-N-(4-[5-fluoro-2-(trifluoromethyl)phenyl]-4-{[4-(trifluoromethyl)phenyl]sulfonyl}cyclohexyl)methanesulfonamide (described in US20110263580); gamma-Secretase Inhibitor I (also known as Z-Leu-Leu-Nle-CHO, benzyloxycarbonyl-leucyl-leucyl-norleucinal, available from Calbiochem); gamma-secretase inhibitor II:

(MOL)(CDX) (available from Calbiochem); gamma secretase inhibitor III, (N-Benzyloxycarbonyl-Leu-leucinal, available from Calbiochem); gamma secretase inhibitor IV, (N-(2-Naphthoyl)-Val-phenylalaninal, available from Calbiochem); gamma-secretase inhibitor V (also known as Z-LF-CHO, N-Benzyloxycarbonyl-Leu-phenylalaninal, available from EMD Millipore); gamma-secretase inhibitor VI (1-(S)-endo-N-(1,3,3)-Trimethylbicyclo[2.2.1]hept-2-yl)-4-fluorophenyl Sulfonamide, available from EMD Millipore); gamma secretase inhibitor VII, (also known as Compound A, MOC-LL-CHO, Menthyloxycarbonyl-LL-CHO, available from Calbiochem); gamma secretase inhibitor X, ({1S-Benzyl-4R-[1-(1S-carbamoyl-2-phenethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2R-hydroxy-5-phenylpentyl}carbamic acid tert-butyl ester, available from Calbiochem); gamma secretase inhibitor XI, (7-Amino-4-chloro-3-methoxyisocoumarin, available from Calbiochem); gamma secretase inhibitor XII, (also known as Z-Ile-Leu-CHO, Shih and Wang, Cancer Res. 67: 1879-1882, 2007); gamma secretase inhibitor XIII, (Z-Tyr-Ile-Leu-CHO, available from Calbiochem); gamma secretase inhibitor XIV, (Z-Cys(t-Bu)-Ile-Leu-CHO, available from Calbiochem); gamma secretase inhibitor XVII, (also known as WPE-III-31C),

(MOL)(CDX) (available from Calbiochem); gamma secretase inhibitor XIX, (also known as benzodiazepine, (2S,3R)-3-(3,4-Difluorophenyl)-2-(4-fluorophenyl)-4-hydroxy-N-((3 S)-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-butyramide, Churcher et al., J Med Chem. 46(12):2275-8, 2003); gamma secretase inhibitor XX, (also known as dibenzazepine, (S,S)-2-[2-(3,5-Difluorophenyl)acetylamino]-N-(5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)propionamide,

(MOL)(CDX) (Weihofen et al., Science 296: 2215-2218, 2002, available from Calbiochem); gamma secretase inhibitor XXI, ((S,S)-2-[2-(3,5-Difluorophenyl)-acetylamino]-N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-propionamide, available from Calbiochem); 5-methyl-2-propan-2-ylcyclohexyl)N-[4-methyl-1-[(4-methyl-1-oxopentan-2-yl)amino]-1-oxopentan-2-yl]carbamate (available from HDH Pharma Inc.); N-trans-3,5-Dimethoxycinnamoyl-Ile-leucinal (available from Calbiochem); N-tert-Butyloxycarbonyl-Gly-Val-Valinal; isovaleryl-V V-Sta-A-Sta-OCH3 (available from Calbiochem); diethyl-(5-phenyl-3H-azepin-2-yl)-amine (described in U.S. Pat. No. 8,188,069); diethyl-(5-isopropyl-3H-azepin-2-yl)-amine (described in U.S. Pat. No. 8,188,069); diethyl-(4-phenyl-3H-azepin-2-yl)-amine (described in U.S. Pat. No. 8,188,069); diethyl-(6-phenyl-3H-azepin-2-yl)-amine (described in U.S. Pat. No. 8,188,069); 5-phenyl-1,3-dihydro-azepin-2-one (described in U.S. Pat. No. 8,188,069); 5-Isopropyl-1,3-dihydro-azepin-2-one (described in U.S. Pat. No. 8,188,069); 4-phenyl-1,3-dihydro-azepin-2-one (described in U.S. Pat. No. 8,188,069); 6-phenyl-1,3-dihydro-azepin-2-one (described in U.S. Pat. No. 8,188,069); 2-butoxy-5-phenyl-3H-azepine (described in U.S. Pat. No. 8,188,069); 1-methyl-5-phenyl-1,3-dihydro-azepin-2-one (described in U.S. Pat. No. 8,188,069); 5-isopropyl-1-methyl-1,3-dihydro-azepin-2-one (described in U.S. Pat. No. 8,188,069); 1-methyl-4-phenyl-1,3-dihydro-azepin-2-one (described in U.S. Pat. No. 8,188,069); 1-methyl-6-phenyl-1,3-dihydro-azepin-2-one (described in U.S. Pat. No. 8,188,069); 1-methyl-5-phenyl-1H-azepine-2,3-dione-3-oxime (described in U.S. Pat. No. 8,188,069); 5-isopropyl-1-methyl-1H-azepine-2,3-dione-3-oxime (described in U.S. Pat. No. 8,188,069); 1-methyl-6-phenyl-1H-azepine-2,3-dione-3-oxime (described in U.S. Pat. No. 8,188,069); 1-methyl-4-phenyl-1H-azepine-2,3-dione-3-oxime (described in U.S. Pat. No. 8,188,069); 3-amino-1-methyl-5-phenyl-1,3-dihydro-azepin-2-one (described in U.S. Pat. No. 8,188,069); 3-amino-5-isopropyl-1-methyl-1,3-dihydro-azepin-2-one (described in U.S. Pat. No. 8,188,069); 3-amino-1-methyl-4-phenyl-1,3-dihydro-azepin-2-one (described in U.S. Pat. No. 8,188,069); 3-amino-1-methyl-6-phenyl-1,3-dihydro-azepin-2-one (described in U.S. Pat. No. 8,188,069); (S)-[1-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-azepin-3-ylcarbamoyl)-ethyl]-carbamic acid tertbutyl ester (described in U.S. Pat. No. 8,188,069); [(S)-1-(5-isopropyl-1-methyl-2-oxo-2,3-dihydro-1H-azepin-3-ylcarbamoyl)-ethyl]carbamic acid tert-butyl ester (described in U.S. Pat. No. 8,188,069); [(S)-1-(1-methyl-2-oxo-4-phenyl-2,3-dihydro-1H-azepin-3-ylcarbamoyl)-ethyl]carbamic acid tert-butyl ester (described in U.S. Pat. No. 8,188,069); [(S)-1-(1-methyl-2-oxo-6-phenyl-2,3-dihydro-1H-azepin-3-ylcarbamoyl)-ethyl]-carbamic acid tert-butyl ester (described in U.S. Pat. No. 8,188,069); (S)-2-amino-N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-azepin-3-yl)-propionamide (described in U.S. Pat. No. 8,188,069); (S)-2-amino-N-(5-isopropyl-1-methyl-2-oxo-2,3-dihydro-1H-azepin-3-yl)propionamide (described in U.S. Pat. No. 8,188,069); (S)-2-Amino-N-(I-methyl-2-oxo-6-phenyl-2,3-dihydro-1H-azepin-3-yl)propionamide hydrochloride (described in U.S. Pat. No. 8,188,069); (S)-2-Amino-N-(I-methyl-2-oxo-4-phenyl-2,3-dihydro-1H-azepin-3-yl)propionamide hydrochloride (described in U.S. Pat. No. 8,188,069); (S)-2-fluoro-3-methyl-butyric acid (described in U.S. Pat. No. 8,188,069); (S)-2-hydroxy-3-methyl-N-[(S)-1-((S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-azepin-3-ylcarbamoyl)-ethyl]-butyramide (described in U.S. Pat. No. 8,188,069); (S)-2-fluoro-3-methyl-N-[(S)-1-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-azepin-3-ylcarbamoyl)-ethyl]-butyramide (described in U.S. Pat. No. 8,188,069); (S)-2-hydroxy-N-[(S)-1-(5-isopropyl-1-methyl-2-oxo-2,3-dihydro-1H-azepin-3-ylcarbamoyl)ethyl]-3-methyl-butyramide (described in U.S. Pat. No. 8,188,069); (S)-2-hydroxy-3-methyl-N-[(S)-1-(1-methyl-2-oxo-4-phenyl-2,3-dihydro-1H-azepin-3-ylcarbamoyl)-ethyl]-butyramide (described in U.S. Pat. No. 8,188,069); (S)-2-hydroxy-3-methyl-N-[(S)-1-(1-methyl-2-oxo-6-phenyl-2,3-dihydro-1H-azepin-3-ylcarbamoyl)-ethyl]-butyramide (described in U.S. Pat. No. 8,188,069); and(S)-2-fluoro-3-methyl-N-[(S)-1-(1-methyl-2-oxo-6-phenyl-2,3-dihydro-1H-azepin-3-ylcarbamoyl)-ethyl]-butyramide (described in U.S. Pat. No. 8,188,069), or pharmaceutically acceptable salts thereof.

Additional examples of gamma-secretase inhibitors are disclosed in U.S. Patent Application Publication Nos. 2004/0029862, 2004/0049038, 2004/0186147, 2005/0215602, 2005/0182111, 2005/0182109, 2005/0143369, 2005/0119293, 2007/0190046, 2008/008316, 2010/0197660 and 2011/0020232; U.S. Pat. Nos. 6,756,511; 6,890,956; 6,984,626; 7,049,296; 7,101,895; 7,138,400; 7,144,910; 7,183,303; 8,188,069; and International Publication Nos. WO 1998/28268; WO 2001/70677, WO 2002/049038, WO 2004/186147, WO 2003/093253, WO 2003/093251, WO 2003/093252, WO 2003/093264, WO 2005/030731, WO 2005/014553, WO 2004/039800, WO 2004/039370, WO 2009/023453, EP 1720909, EP 2178844, EP 2244713.

The entire disclosures of all of the foregoing are hereby incorporated by reference herein.

Atoh1 Stimulatory Compounds

Compounds that promote progenitor cell differentiation to Atoh1+ hair cells include one or more of CHIR99021, LY411575, vorinostat, MEEI-0000489, MEEI-0087336, MEEI-0007991, 1-Azakenpaullone, BIO, WAY-262611, NP031112, MG-132, IM-12, Trichostatin A, HLY78, and PF03084014. In some embodiments, a combination of CHIR99021 and LY411575 is used.

GSK-3-Beta Inhibitors

GSK3β inhibitors include, but are not limited to, lithium chloride (LiCl), Purvalanol A, olomoucine, alsterpaullone, kenpaullone, benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3 inhibitor II), 2,4-dibenzyl-5-oxothiadiazolidine-3-thione (OTDZT), (2′Z,3′E)-6-Bromoindirubin-3′-oxime (BIO), α-4-Dibromoacetophenone (i.e., Tau Protein Kinase I (TPK I) Inhibitor), 2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone, N-(4-Methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418), and indirubins (e.g., indirubin-5-sulfonamide; indirubin-5-sulfonic acid (2-hydroxyethyl)-amide indirubin-3′-monoxime; 5-iodo-indirubin-3′-monoxime; 5-fluoroindirubin; 5,5′-dibromoindirubin; 5-nitroindirubin; 5-chloroindirubin; 5-methylindirubin, 5-bromoindirubin), 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3 inhibitor II), 2,4-Dibenzyl-5-oxothiadiazolidine-3-thione (OTDZT), (2′Z,3′E)-6-Bromoindirubin-3′-oxime (BIO), α-4-Dibromoacetophenone (i.e., Tau Protein Kinase I (TPK I) Inhibitor), 2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone, (vi) N-(4-Methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418), and H-KEAPPAPPQSpP-NH2 (L803) or its cell-permeable derivative Myr-N-GKEAPPAPPQSpP-NH2 (L803-mts). Other GSK3β inhibitors are disclosed in U.S. Pat. Nos. 6,417,185; 6,489,344; 6,608,063 and Published U.S. Applications Nos. 690497, filed Oct. 20, 2003; 468605, filed Aug. 19, 2003; 646625, filed Aug. 21, 2003; 360535, filed Feb. 6, 2003; 447031, filed May 28, 2003; and 309535 filed Dec. 3, 2002. In some embodiments, the methods include administration of a CK1 inhibitor (e.g., a small molecule CK1 inhibitor) and a GSK3β inhibitor, plus one or both of a gamma-secretase inhibitor and/or an Atoh1 stimulatory compound as described herein.

Methods of Treatment

The combinations and methods described herein are appropriate for the treatment of mammalian (e.g., human) subjects who have or are at risk of developing hearing disorders resulting from cochlear hair cell loss. In some embodiments the subjects are post-neonatal (e.g., child, adolescent or adult, e.g., above the age of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 years) subjects. The methods described herein can be used to treat cochlear hair cell loss and any disorder that arises as a consequence of hair cell loss in the ear, such as hearing impairments or deafness. These subjects can receive treatment with a combination of agents as described herein. The approach may be optimal for treatment of acute hearing loss shortly after the damage has occurred, and may be less effective after longer time periods when Notch signaling has returned to its baseline level in the adult.

In some instances, methods include selecting a subject. Subjects suitable for treatment include those at risk of hair cell loss or with hair cell loss and/or those at risk of sensorineural hearing loss or with sensorineural hearing loss. Any subject experiencing or at risk for developing hearing loss is a candidate for the treatment methods described herein. A human subject having or at risk for developing a hearing loss can hear less well than the average human being, or less well than a human before experiencing the hearing loss. For example, hearing can be diminished by at least 5, 10, 30, 50% or more.

The subject can have hearing loss associated with cochlear hair cell loss for any reason, or as a result of any type of event. For example, a subject can be deaf or hard-of-hearing as a result of an infection or physical ototoxic insult, e.g., a traumatic event, such as a physical trauma to a structure of the ear that does not irreversibly damage the supporting cells. In preferred embodiments, the subject can have (or be at risk of developing) hearing loss as result of exposure to a sudden loud noise, or a prolonged exposure to loud noises. For example, prolonged or repeated exposures to concert venues, airport runways, and construction areas can cause inner ear damage and subsequent hearing loss; subjects who are subjected to high levels of environmental noise, e.g., in the home or workplace, can be treated using the methods described herein. A subject can have a hearing disorder that results from aging, e.g., presbycusis, which is generally associated with normal aging processes; see, e.g., Huang, Minn. Med. 90(10):48-50 (2007) and Frisina, Annals of the New York Academy of Sciences, 1170: 708-717 (2009), and can occur in subjects as young as 18, but is generally more marked in older subjects, e.g., subjects over age 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90. A subject can have tinnitus (characterized by ringing in the ears) due to loss of hair cells. A subject can experience a chemical ototoxic insult, wherein ototoxins include therapeutic drugs including antineoplastic agents, salicylates, quinines, and aminoglycoside antibiotics, e.g., as described further below, contaminants in foods or medicinals, and environmental or industrial pollutants. In general, subjects who have a known genetic disease associated with hearing loss (e.g., mutations in connexin 26, Alport, and so on), or a known cause of hearing loss that is associated with structural damage to the inner ear (e.g. penetrating trauma), that would not be correctable or ameliorated by the present methods are excluded from the present methods. In some embodiments, subjects who lack supporting cells, e.g., who have no LGR5+ cells in their cochlea, are excluded from treatment, or are administered LGR5+ cells as part of the treatment.

In some embodiments, the methods include administering to the subject a compound described herein within one, two, three, four, five, six, or seven days, or one, two, three, four, five, or six weeks of exposure to an ototoxic insult, e.g., a physical (noise, trauma) or chemical (ototoxin) insult that results in or could result in a loss of hair cells, and causes an increase in Notch signaling in the subject.

In some embodiments, a subject suitable for the treatment using the compounds and methods featured in the invention can include a subject having a vestibular dysfunction, including bilateral and unilateral vestibular dysfunction; the methods include administering a therapeutically effective amount of an agent described herein, e.g., by systemic administration or administration via the endolymphatic sac (ES). Vestibular dysfunction is an inner ear dysfunction characterized by symptoms that include dizziness, imbalance, vertigo, nausea, and fuzzy vision and may be accompanied by hearing problems, fatigue and changes in cognitive functioning. Vestibular dysfunctions that can be treated by the methods described herein can be the result of a genetic or congenital defect; an infection, such as a viral or bacterial infection; or an injury, such as a traumatic or nontraumatic injury, that results in a loss of vestibular hair cells. In some embodiments, balance disorders or Meniere's disease (idiopathic endolymphatic hydrops) may be treated by the methods described herein. Vestibular dysfunction is most commonly tested by measuring individual symptoms of the disorder (e.g., vertigo, nausea, and fuzzy vision).

Alternatively or in addition, the compounds and methods featured in the invention can be used prophylactically, such as to prevent, reduce or delay progression of hearing loss, deafness, or other auditory disorders associated with loss of hair cells. For example, a composition containing one or more compounds can be administered with (e.g., before, after or concurrently with) an ototoxic therapy, i.e., a therapeutic that has a risk of hair cell toxicity and thus a risk of causing a hearing disorder. Ototoxic drugs include the antibiotics neomycin, kanamycin, amikacin, viomycin, gentamycin, tobramycin, erythromycin, vancomycin, and streptomycin; chemotherapeutics such as cisplatin; nonsteroidal anti-inflammatory drugs (NSAIDs) such as choline magnesium trisalicylate, diclofenac, diflunisal, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, salsalate, sulindac, and tolmetin; diuretics; salicylates such as aspirin; and certain malaria treatments such as quinine and chloroquine. For example, a subject undergoing chemotherapy can be treated using the compounds and methods described herein. The chemotherapeutic agent cisplatin, for example, is known to cause hearing loss. Therefore, a composition containing one or more compounds can be administered with cisplatin therapy (e.g., before, after or concurrently with) to prevent or lessen the severity of the cisplatin side effect. Such a composition can be administered before, after and/or simultaneously with the second therapeutic agent. The two agents may be administered by different routes of administration.

In general, the compounds and methods described herein can be used to generate hair cell growth in the ear and/or to increase the number of hair cells in the ear (e.g., in the inner, middle, and/or outer ear). For example, the number of hair cells in the ear can be increased about 2-, 3-, 4-, 6-, 8-, or 10-fold, or more, as compared to the number of hair cells before treatment. This new hair cell growth can effectively restore or establish at least a partial improvement in the subject's ability to hear. For example, administration of an agent can improve hearing loss by about 5, 10, 15, 20, 40, 60, 80, 100% or more.

In some instances, compositions can be administered to a subject, e.g., a subject identified as being in need of treatment, using a systemic route of administration. Systemic routes of administration can include, but are not limited to, parenteral routes of administration, e.g., intravenous injection, intramuscular injection, and intraperitoneal injection; enteral routes of administration e.g., administration by the oral route, lozenges, compressed tablets, pills, tablets, capsules, drops (e.g., ear drops), syrups, suspensions and emulsions; transdermal routes of administration; and inhalation (e.g., nasal sprays).

In some instances, compositions can be administered to a subject, e.g., a subject identified as being in need of treatment, using a systemic or local route of administration. Such local routes of administration include administering one or more compounds into the ear of a subject and/or the inner ear of a subject, for example, by injection and/or using a pump.

In some instances, compositions can be can be injected into the ear (e.g., auricular administration), such as into the luminae of the cochlea (e.g., the Scala media, Sc vestibulae, and Sc tympani). For example, compositions can be administered by intratympanic injection (e.g., into the middle ear), intralabyrinthine delivery (e.g., to the stapes foot plate), and/or injections into the outer, middle, and/or inner ear. Such methods are routinely used in the art, for example, for the administration of steroids and antibiotics into human ears. Injection can be, for example, through the round window of the ear or through the cochlea capsule. In another exemplary mode of administration, compositions can be administered in situ, via a catheter or pump. A catheter or pump can, for example, direct a pharmaceutical composition into the cochlea luminae or the round window of the ear. Exemplary drug delivery apparatus and methods suitable for administering one or more compounds into an ear, e.g., a human ear, are described by McKenna et al., (U.S. Publication No. 2006/0030837) and Jacobsen et al., (U.S. Pat. No. 7,206,639). In some embodiments, a catheter or pump can be positioned, e.g., in the ear (e.g., the outer, middle, and/or inner ear) of a subject during a surgical procedure. In some embodiments, a catheter or pump can be positioned, e.g., in the ear (e.g., the outer, middle, and/or inner ear) of a subject without the need for a surgical procedure.

In some instances, compositions can be administered in combination with a mechanical device such as a cochlea implant or a hearing aid, which is worn in the outer ear. An exemplary cochlea implant that is suitable for use with the present invention is described by Edge et al., (U.S. Publication No. 2007/0093878).

In some instances, compositions can be administered according to any of the Food and Drug Administration approved methods, for example, as described in CDER Data Standards Manual, version number 004 (which is available at fda.give/cder/dsm/DRG/drg00301.htm).

In some instances, the present disclosure includes treating a subject by administering to the subject cells produced using the compositions and methods disclosed herein. In general, such methods can be used to promote complete or partial differentiation of a cell to or towards a mature cell type of the inner ear (e.g., a hair cell) in vitro. Cells resulting from such methods can then be transplanted or implanted into a subject in need of such treatment. Cell culture methods required to practice these methods, including methods for identifying and selecting suitable cell types, methods for promoting complete or partial differentiation of selected cells, methods for identifying complete or partially differentiated cell types, and methods for implanting complete or partially differentiated cells are described herein. Target cells suitable for use in these methods are described above.

In some instances, methods can include administering one or more compositions disclosed herein and cells produced using the compositions and methods disclosed herein to a subject.

Administration of cells to a subject, whether alone or in combination with compounds or compositions disclosed herein, can include administration of undifferentiated, partially differentiated, and fully differentiated cells, including mixtures of undifferentiated, partially differentiated, and fully differentiated cells. As disclosed herein, less than fully differentiated cells can continue to differentiate into fully differentiated cells following administration to the subject.

Where appropriate, following treatment, the subject can be tested for an improvement in hearing or in other symptoms related to inner ear disorders. Methods for measuring hearing are well-known and include pure tone audiometry, air conduction, and bone conduction tests. These exams measure the limits of loudness (intensity) and pitch (frequency) that a subject can hear. Hearing tests in humans include behavioral observation audiometry (for infants to seven months), visual reinforcement orientation audiometry (for children 7 months to 3 years); play audiometry for children older than 3 years; and standard audiometric tests for older children and adults, e.g., whispered speech, pure tone audiometry; tuning fork tests; brain stem auditory evoked response (BAER) testing or auditory brain stem evoked potential (ABEP) testing. Oto-acoustic emission testing can be used to test the functioning of the cochlear hair cells, and electro-cochleography provides information about the functioning of the cochlea and the first part of the nerve pathway to the brain. In some embodiments, treatment can be continued with or without modification or can be stopped.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1. CK1 Destabilizes Atoh1

We performed immunoprecipitation mass spectrometry (IP-MS) to identify binding partners of Atoh1 using a stably expressing 293T cell line prepared by lentiviral infection of pHAGE-FLAG-HA-Atoh1¹. Lysates of FLAG-HA-Atoh1 293T cells immunoprecipitated with HA antibody were subjected to mass spectrometry Two isoforms of casein kinase, CK1δ and ε were found associated with Atoh1 (Table I).

Atoh1-CK1 association was further validated by co-immunoprecipitation confirming that CK1 binds to Atoh1 (FIG. 1). We also found that CK1 overexpression decreased steady-state level of Atoh1.

We previously found that Huwe1, an E3 ubiquitin ligase, directed Atoh1 degradation to the proteasome (see Cheng, “Role of the ubiquitin-proteasome pathway in the inner ear: identification of an E3 ubiquitin ligase for Atoh1,” Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2014, available online at hdl.handle.net/1721.1/96458; and PCT/US2015/043976). To explore the effect of CK1 on proteasomal degradation of Atoh1, we performed co-IP experiments. We found that two small molecule CK1 inhibitors, D4476 and PF-670462, diminished Atoh1-Huwe1 binding and subsequent ubiquitylation (FIG. 2).

TABLE I Mass spectrometric analysis to confirm immunoprecipitation of Atoh1 and CK1 Average Gene Total Unique peptide Protein symbol peptide peptide score coverage TUBB2A 159 40 3.0903 55.51% ATOH1 155 19 2.1144 34.18% TUBA1A 112 34 3.1383 66.08% RPL4 106 36 2.4767 53.63% EEF1A1 103 28 2.6454 58.87% TUFM 72 38 3.2781 68.36% RPL3 67 29 2.5399 45.66% SSB 65 30 2.8608  5.43% RBMX 65 27 2.5009 47.06% ENO1 62 34 3.1674 58.29% PSMC5 52 31 3.5295 64.29% PSMC2 50 29 2.9925 63.74% YARS2 11 8 3.3677 29.77% TUBA1C 7 11 3.6062 15.14% SERPINH1 9 7 2.5123 20.81% CSNK1D 7 4 3.1615  12.5% CSNK1E 6 3 2.8233  9.86% ACOX1 5 4 2.8799 10.30% BCKDHA 5 3 2.6629  9.44% PRPS1 3 3 2.6642 18.46% TUBB2B 2 1 3.0318  2.70% TUBA4A 1 1 3.4258 31.31% PRDX4 1 1 3.1432  7.01% AAMP 1 1 2.3377  2.53% PDE4B 1 1 2.2331  2.04% Coomassie blue stained bands were excised and subjected to mass spectrometric analysis after immunoprecipitation of Atoh1 and its associated binding partners.

Example 2. Evolutionarily Conserved Serines in the C-Terminus Account for Atoh1 Stability

We generated a panel of deletions of Atoh1, retaining the bHLH domain (FIG. 3A, right panel). Of two N-terminal (Δ10-93 for deletion 1 and Δ94-156 for deletion 2) and two C-terminal (Δ214-305 for deletion 3 and Δ306-347 deletion 4) deletions, Atoh1-deletion 4 had the longest half-life based on a cycloheximide chase assay, suggesting that motifs affecting the half-life of Atoh1 fell between amino acids 306 and 347 (FIGS. 3A-B).

Deletion in the serine-enriched C-terminal motif diminished Huwe1 binding and subsequent ubiquitylation, indicating that sequences affecting involved in E3 ubiquitin ligase binding and enzymatic activity toward Atoh1 may lie in this area (FIG. 4).

Cross-species sequence comparison of Atoh1 by MegaAlign (DNAstar, Madison, Wis.) indicated that serines 309, 325, 328, 331 and 334 were conserved across species (FIG. 5A). Since conservation may relate to biological function, we generated mutated Atoh1 plasmids containing alanine in the place of each serine. The S334A mutant was protected from degradation based on its higher level of expression and the lack of any further effect of proteasome inhibition with MG132, while other mutants were affected to a similar extent as wild-type Atoh1 (FIG. 5B). Mutations at positions 328 and 331 had modest effects, while mutation at position 334 dramatically prolonged the half-life of Atoh1 (FIG. 5C). Co-immunoprecipitation also showed that 5334 mutation affected Huwe1 binding and ubiquitylation (FIG. 6). We conclude that Ser 334 in the C-terminus of Atoh1 contains a motif (“degron”) that specifies Atoh1 for proteasomal degradation.

Example 3. Mass Spectrometry Identifies Atoh1 Phosphorylation in the Presence of CK1 Overexpression

Atoh1 phosphorylation sites controlled by CK1, Mass spectrometry analysis of immunoprecipitated Atoh1 from FLAG-Atoh1 plasmids in HEK cells with or without CK1 showed that, among the conserved serine sites on the C-terminus of Atoh1, Serine 325, 328 and 334 were phosphorylated after CK1 overexpression (Table II). These data indicate that Atoh1 phosphorylation at S334 is critical for CK1-mediated Atoh1 degradation.

TABLE II Summary of Atoh1 Phosphorylation Atoh1 + Conser- Contribution SEQ ID Position^(a) Atohl^(b) CK1^(c) vation^(d) to stability^(e) Peptide^(f) NO:  82 x x Y N/A AAQYLLHSPELGASEAAAPR  1  99 x x N N/A DEADSQGELVR  2 309 x x N No DLSPSLPGGILQPVQEDNSK  3 311 x N** N/A DLSPSLPGGILQPVQEDNSK  4 325 x Y No DLSPSLPGGILQPVQEDNSKTSPR  5 328 x Y Low DLSPSLPGGILQPVQEDNSKTSPR  6 331 x Y Low SHRSDGEFSPHSHYSDSDEAS  7 334 x Y High SDGEFSPHSHYSDSDEAS  8 339 x N* N/A SDGEFS#PHSHYSDSDEAS  9 345 X N* N/A SDGEFSPHSHYSDSDEAS 10 347 x N* N/A SDGEFSPHSHYSDSDEAS 11 ^(a)Position of the amino acids ^(b)Atoh1 overexpression only ^(c)Atoh1 and CK1 overexpression ^(d)Cross-species sequence comparison. Y: conserved, N*: non-conserved in one tested species, N**: non-conserved in two tested species, N: non-conserved ^(e)Cycloheximide-chase assay results shown in FIG. 5 ^(f)Phosphorylated peptide sequence: phosphorylation is underlined

Example 4. CK1 Regulates Downstream Signaling by Atoh1

To further assess potential roles of CK1 in the control of Atoh1 signaling, we performed dual luciferase reporter assays using a firefly reporter construct with an Atoh1 E-box associated motif (AtEAM) and a Renilla control reporter. AtEAM is a ten amino acid Atoh1-specific binding motif that represents the site for the activity of Atoh1 in numerous downstream genes (Klisch et al., Proc Natl Acad Sci USA. 2011 Feb. 22; 108(8):3288-93). Inhibition of CK1 by D4476 increased and overexpression of CK1 (specifically CK1δ and/or CK1c) reduced Atoh1 downstream signaling (FIG. 7A).

We then compared the effects on E-box luciferase activity of wild-type Atoh1 vs the serine mutants at the conserved Atoh1 C-terminus after overexpression of CK1δ and CK1ε (FIG. 7B). CK1 overexpression caused a significant decrease in downstream signaling in S325A, S328A, S331A and S339A, but not wild-type or S334A Atoh1 (FIG. 7C), supporting a role of CK1 mediated S334 phosphorylation in cellular Atoh1 activity.

Example 5. CK1 Inhibition Increased Hair Cell Generation in the Organ of Corti

Treatment of organ of Corti explants from newborn mice with 10 μM CK1 inhibitor D4476 for 72 hours caused stabilization of Atoh1 protein in the cochlea based on densitometry (FIG. 8).

Since CK1 inhibition stabilized Atoh1, we assessed its effect on hair cell generation in the cochlea. A Sox2-positive Cre-reporter strain crossed with CAG-TdTomato (Bramhall et al., Stem cell reports 2, 311-322 (2014)) was used to label all supporting cells for lineage tracing to follow supporting cells after CK1 inhibition. Treatment of organ of Corti explants with 10 μM CK1 inhibitor D4476 for 72 hours significantly increased the number of reporter-labeled outer and inner hair cells in all regions of cochlea (FIG. 9), indicating that the hair cells made after CK1 inhibition came from Sox2-positive supporting cells.

Example 6. CK1 Inhibition Promoted Hair Cell Regeneration after Aminoglycoside Damage

Aminoglycoside-exposed organ of Corti explants³ were treated with 10 μM D4476 for 72 hours and lineage traced. The treatment resulted in an increase of Sox2-lineage-tagged hair cells (cells co-labeled with myosin VIIa and TdTomato) indicating that Atoh1 stabilization by CK1 inhibition regenerated hair cells (FIG. 10).

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. 

What is claimed is:
 1. A method of treating a subject who has or is at risk of developing hearing loss or vestibular dysfunction, comprising administering to the subject one or more inhibitory nucleic acids that target casein kinase 1 (CK1) epsilon and/or CK1 delta, and one or more compounds that stimulate Atoh1 gene expression.
 2. The method of claim 1, wherein the subject has or is at risk for developing sensorineural hearing loss, auditory neuropathy, or both.
 3. The method of claim 1, wherein the subject has or is at risk for developing a vestibular dysfunction that results in dizziness, imbalance, or vertigo.
 4. The method of claim 1, wherein the one or more inhibitory nucleic acids that target CK1 epsilon and/or CK1 delta, and/or the one or more compounds that stimulate Atoh1 gene expression is administered systemically.
 5. The method of claim 1, wherein the one or more inhibitory nucleic acids that target CK1 epsilon and/or CK1 delta, and/or the one or more compounds that stimulate Atoh1 gene expression is administered locally to the inner ear.
 6. The method of claim 1, wherein the one or more inhibitory nucleic acids that target CK1 epsilon and/or CK1 delta is shRNA or siRNA.
 7. The method of claim 1, wherein the method comprises administering to the subject one or more inhibitory nucleic acids that target CK1 epsilon.
 8. The method of claim 1, wherein the method comprises administering to the subject one or more inhibitory nucleic acids that target CK1 delta.
 9. The method of claim 1, wherein the one or more compounds that stimulate Atoh1 gene expression comprises a glycogen synthase kinase 3 beta (GSK-3-beta) inhibitor.
 10. The method of claim 1, wherein the one or more compounds that stimulate Atoh1 gene expression comprises a gamma secretase inhibitor.
 11. The method of claim 1, wherein the hearing loss or vestibular dysfunction is caused by aging.
 12. The method of claim 1, wherein the hearing loss or vestibular dysfunction is caused by a genetic or congenital defect.
 13. The method of claim 1, wherein the hearing loss or vestibular dysfunction is caused by noise or trauma.
 14. The method of claim 1, wherein the hearing loss or vestibular dysfunction is caused by a chemical ototoxic insult. 